Bile acids are important physiological agents that are required for the disposal of cholesterol and the absorption of dietary lipids and lipid soluble vitamins. Bile salts are the water-soluble end products of cholesterol, and are synthesized de novo in the liver. During normal enterohepatic circulation (EHC), the average bile salt pool is secreted into the duodenum twice during each meal, or an average of 6-8 times per day for the purpose of forming mixed micelles with the products of lipid digestion. During intestinal transit, most of the secreted bile salt is absorbed in the terminal ileum and is returned to the liver via the portal vein. The bile salt pool is replenished by hepatic synthesis of new bile from serum cholesterol. It has been shown that upon surgical, pharmacological or pathological interruption of the EHC, bile salt synthesis is increased up to 15-fold, leading to an increased demand for cholesterol in the liver. Therefore, various studies have been reported suggesting possible oral bacterial preparations for reducing serum cholesterol. Though effective, these methods still have several limitations. For example, a normal daily intake of 250 ml of yogurt would only correspond to 500 milligram of cell dry weight (CDW) of bacteria, and of those bacteria ingested only 1% would survive gastric transit limiting the overall therapeutic effect. There are also some practical concerns regarding the production, cost, and storage of such a product (De Smet et al., 1998). Further, oral administration of live bacterial cells can pose problems. For example, when given orally, large amounts of live bacterial cells can stimulate host immune response, they can be retained in the intestine, and repeated large doses could result in their replacing the normal intestinal flora (De Boever and Verstraete, 1999; Christiaens et al., 1992). In addition, risk of systemic infections, deleterious metabolic activities, adjuvant side-effects, immuno-modulation and risk of gene transfer has limited their use (De Boever and Verstraete, 1999; Christiaens et al., 1992). Metabolic activities and immuno-modulation, have limited its clinical use (De Boever et al., 2000).
Although bile acids are important to normal human physiology, bile acids can be cytotoxic agents when produced in pathologically high concentrations. As well, when ileal transport of bile acids is defective due to a congenital defect, resection of the ileum, or disease, elevated intraluminal concentrations of bile acids can induce the secretion of electrolytes and water causing diarrhea and dehydration. Therefore, various studies suggested methods for removing bile acids by either directly preventing the reabsorption of bile acids or by removing bile acids using chemical binders such as bile acid sequestrants (BAS). These methods have several limitations. For example, common BAS Cholestyramine resin (Locholest, Questran), Colesevelam (Welchol), and Colestipol (Colestid) are well documented to exhibit major adverse effects such as nausea, bloating, constipation, and flatulence (Christiaens et al., 1992).
Current treatments for elevated blood cholesterol include dietary management, regular exercise, and drug therapy with fibrates, bile acid sequestrants, and statins. Such therapies are often sub-optimal and carry a risk for serious side effects. Dietary intervention, whereby lipid intake is restricted is generally the first line of treatment (Lichtenstein, 1998; Ornish and Denke, 1994; Ornish et al., 1998). Studies show that complete elimination of dietary cholesterol and limiting fat content to less than ten percent of the daily caloric intake can effect a mere four percent regression of atherosclerotic plaques after five years when combined with stress management and aerobic exercise (Dunn-Emke et al., 2001). However, the combined restricted vegetarian diet (free of meat, fish, chicken, vegetable oils and all dairy fat products) and aerobic approach, is unrealistic for all but the most dedicated individuals. A variety of dietary supplements or specific foods e.g. brans, psylliums, guar gum, lecithins, whey, red wines, fish oils and ginseng root extract have been reported to reduce high blood cholesterol or its consequences. The mechanisms are varied and include cholesterol sequestering, chelating, entrapment and oxidation inhibition. Such regimens generally lower the blood cholesterol level by ten percent or less. In addition, none of these dietary interventions have been shown to arrest or cure atherosclerosis or other high blood cholesterol associated diseases.
Pharmacologic agents such as fibric acid derivatives (fibrates), nicotinic acid, bile acid sequestrants (BAS), estrogen replacement therapy, and hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are also available for the treatment of high cholesterol. From among the agents listed above, the statins are considered to have the most potential for treatment. Currently lovastatin, pravastatin, zocor, fluvastatin and atorvastatin are being used for clinical lowering of cholesterol. Although effective at reducing cholesterol levels, they are nevertheless expensive (Attanasio et al., 2001; Hodgson and Cohen, 1999; Prosser et al. 2000; Reckless, 1996). Some are known to have side effects and are associated. Naturally occurring bacteria can significantly lower serum cholesterol levels by hydrolyzing bile salts in the intestinal tract but only 1% of free bacteria ingested survive the GI transit. However, live bacterial cells can cause a host immune response and can be retained in the intestine replacing the natural intestinal flora (Taranto et al., 2000; Anderson and Gilliland, 1999; Chin et al., 2000). It has been shown that certain strains of bacteria act directly on bile acids in the gastrointestinal tract and may be beneficial in reducing serum cholesterol levels in this way (Taranto et al., 2000; Anderson and Gilliland, 1999; De Smet et al. 1994). Control of cholesterol through oral live bacterial cell therapy, is based on the demonstration that naturally occurring bacteria such as Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus reuteri can significantly lower serum cholesterol levels (Taranto et al., 2000; Anderson and Gilliland, 1999; De Smet et al. 1994). For example, Lactobacillus reuteri was used to decrease the serum cholesterol in pigs through interaction of free bacteria with the host's bile salt metabolism (De Smet et al., 1998). The underlying mechanism for the reduction of serum cholesterol appears to be the capacity of Lactobacillus to hydrolyze bile salts in the intestinal tract (Anderson and Gilliland, 1999; De Smet et al. 1994). Elevated Bile Salt Hydrolase (BSH) activity leads to an increase in the loss of bile acids from the ECH and to a greater demand for cholesterol by the liver (De Smet et al. 1994) (FIG. 8). In the work of De Smet et al., the BSH activity of BSH overproducing. Lactobacillus plantarum 80 (pCBHl) was shown to have a considerable cholesterol lowering capacity (De Smet et al. 1994). The bile salt hydrolase enzyme, contained on the multicopy plasmid (pCBHl), carries out the deconjugation of bile salts through catalysis of hydrolysis of the amide bond that conjugates bile acids to glycine or taurine (Christiaens et al., 1992; De Smet et al. 1994) (FIG. 9).
While work in this field has been very promising, several limiting factors to the oral administration of free bacteria have been identified. The therapeutic potential of free bacteria is hampered by inherent limitations in their use. For example, of those free bacteria ingested only 1% survive gastric transit limiting the overall therapeutic effect (De Smet et al. 1994). Also, oral administration of live bacterial cells can cause a host immune response, and can be detrimentally retained in the intestine replacing the natural intestinal flora (Taranto et al., 2000; Chin et al., 2000; De Boever and Verstraete, 1999). Furthermore, there are some practical concerns regarding the production, cost, and storage of products containing free bacteria (De Boever and Verstraete, 1999). Thus, concerns of safety and practicality have prevented the regular use of this promising therapy in clinical practice.
Other problematic diseases or disorders arise from disrupted lipid metabolism. For example, steathorrea results from damage to the pancreas or bowel (e.g. inflammation resulting from pancreatitis). The pancreas is the gland that produces digestive enzymes to metabolize carbohydrates and lipids. The resulting condition, known as exocrine or pancreatic insufficiency, leads to weight loss and very foul-smelling stools or diarrhea. Chronic pancreatitis can lead to diabetes and pancreatic calcification, a condition where small, hard deposits form in the pancreas. There is a need for new treatments that allow patients to fully digest food.
Encapsulation and immobilization patents include U.S. Pat. No. 6,565,777, U.S. Pat. No. 6,346,262, U.S. Pat. No. 6,258,870, U.S. Pat. No. 6,264,941, U.S. Pat. No. 6,217,859, U.S. Pat. No. 5,766,907 and U.S. Pat. No. 5,175,093. Artificial cell microencapsulation is a technique used to encapsulate biologically active materials in specialized ultra thin semi-permeable polymer membranes (Chang and Prakash, 1997; Chang, 1964). The polymer membrane protects encapsulated materials from harsh external environments, while at the same time allowing for the metabolism of selected solutes capable of passing into and out of the microcapsule. In this manner, the enclosed material is retained inside and separated from the external environment, making microencapsulation particularly useful for biomedical and clinical applications (Lim and Sun, 1980; Sefton et al, 2000; Chang, 1999). Studies show that artificial cell microcapsules can be used for oral administration of live genetically engineered cells that can be useful for therapeutic functions (Prakash and Chang, 2000; Prakash and Chang, 1996). Examples of applications of microencapsulation of enzymes, cells and genetically engineered microorganisms are xanthine oxidase for Lesch-Nyhan disease; phenylalanine ammonia lyase for pheny, ketonuria and E. coli DH5 cells for lowering urea, ammonia and other metabolites (Chang and Prakash 2001). Although the live cells remain immobilized inside the microcapsules, microencapsulation does not appear to hinder their growth kinetics (Prakash and Chang, 1999). The microcapsules remain intact during passage through the intestinal tract and are excreted intact with the stool in about 24 hours. The cells are retained inside, and excreted with, the intact microcapsules addressing many of the major safety concerns associated with the use of live bacterial cells for various clinical applications. The membranes of the microcapsules are permeable to smaller molecules, and thus the cells inside the microcapsules metabolize small molecules found within the gut during passage through the intestine (Chang and Prakash, 1997; Prakash and Chang, 2000; Prakash and Chang, 1996; Prakash and Chang, 1999; Prakash and Chang, 1996a, Prakash and Chang, 1999a).